Project summary: Synthesis of mRNA in eukaryotes, and its utilization in the cytoplasm, requires modification at the RNA's 3' end by addition of a poly(A) tail. This process also serves as a point at which the cell can regulate the type and amount of mRNA derived from a particular gene. Even though mRNA 3' end formation occurs in an unexpectedly large complex, most, if not all, of the subunits of this machinery have been identified in the yeast S. cerevisiae. However, little is known about how these 21 proteins cooperate with each other to insure processing that is accurate as well as coupled in a timely fashion to other events in mRNA synthesis and packaging. With the 3'-end processing components in hand, and activities for several of these factors newly defined, a unique opportunity now exists to rigorously address the mechanism by which this essential and universal step in gene expression occurs and is regulated. The central hypothesis of this proposal is that definable rearrangements of protein partners occur within the complex as it evaluates the authenticity of the processing site, commits to cleavage at the poly(A) site, reorganizes to position the poly(A) polymerase for tail synthesis, and releases the final RNA product. The objective is to understand how such reorganizations drive the cycle of mRNA 3' end processing. The research focuses on four events that are likely to be critical transition points in this cycle yet whose underlying mechanisms are not understood. These include the initiation of cleavage and the role of the Ssuy2 protein in this step, the transition from cleavage to poly(A) addition and its regulation by phosphorylation of the Ptai scaffold protein, the control of poly(A) polymerase activity by interactions at its amino-terminus, and the release of processing factors following tail synthesis. The studies will use yeast as the model organism because of the ease of introducing tags and mutations into the genome, the availability of numerous 3' end processing mutants, the ease of purifying processing factors for biochemical studies, and the high degree of conservation of the 3' end processing machinery across eukaryotes. Relevance: Without poly(A) tails, mRNA is targeted for degradation in the nucleus, it does not get out of the nucleus very well, and it is not translated efficiently or turned over at the appropriate rate in the cytoplasm. Maturation of mRNA 3' ends is functionally linked to other essential processes such as transcription, mRNA export, chromosome segregation, DNA repair, and tissue-specific protein expression. Mistakes in polyadenylation can impact on all of these processes. The proposed research should significantly advance our insight into the dynamics of this essential step in mRNA synthesis and identify points at which the constitutive process is likely to be regulated. [unreadable] [unreadable] [unreadable]